Enterohemorrhagic E. coli (EHEC) O157:H7 causes bloody diarrhea and hemolytic uremic syndrome (HUS) throughout the world. EHEC has a very low infectious dose, making it difficult to control epidemiologically. EHEC colonizes the large intestine where it causes attaching and effacing (AE) lesions, and also produces Shiga toxins (Stx) that are responsible for the major symptoms of HUS. We recently reported that EHEC senses three signals to activate transcription of virulence genes: a bacterial aromatic autoinducer (AI-3) produced by the normal gastrointestinal flora, and the hormones epinephrine/norepinephrine produced by the host. These signals are detected by membrane bound sensor kinases that subsequently relay this information to a complex regulatory cascade to activate transcription of virulence genes. We have identified several members of this signaling cascade: the QseBC and QseEF two-component systems, and the QseA and QseD LysR-like regulators. However, many aspects of this regulatory cascade, which are crucial for EHEC virulence, remain poorly understood. This proposal represents a comprehensive effort to study the AI-3/epinephrine/norepinephrine signaling cascade, responsible for virulence gene regulation in EHEC. In Specific Aim 1, we will perform a detailed characterization of the QseBC regulon to map this complex regulatory network. In Specific Aim 2, we will characterize the protein-protein interactions between the QseA and QseD LysR-like regulators and their effect on gene regulation. Specific Aim 3 is designed to characterize the role of the novel QseEF two-component system in EHEC pathogenesis. The proposed experimental approaches will achieve a better understanding of the sensory events by which EHEC responds to activate its virulence genes, and may lead to the identification of other virulence genes and novel targets for the potential development of more effective intervention strategies for EHEC disease.